Periventricular leukomalacia (PVL) is the principal white matter lesion underlying cerebral palsy and a leading cause of chronic neurological deficits in survivors of premature birth. Inflammation associated with ischemia/hypoxia and maternal/fetal infection is a major determinant of the pathogenesis of PVL, with characteristic astrogliosis and microglial activation in the cerebral white matter. However, the mechanism of selective injury to premyelinating oligodendrocytes (pre-OLs) under these inflammatory conditions is poorly understood. Our long-term goal is to determine the molecular basis of pre-OL injury in order to develop strategies to prevent PVL. Recently, we showed that microglia, activated by lipopolysaccaride (LPS), selectively kill pre-OLs by producing peroxynitrite. Interestingly, although astrocytes are not required for LPS-induced toxicity, their presence switches the activated microglial toxicity from a peroxynitrite- dependent mechanism to a mechanism dependent upon the proinflammatory cytokine, tumor necrosis factor 1 (TNF1). Exposure to TNF1 results in pre-OL death in mixed glial cultures but had only minimal effect to purified pre-OLs. Non-injurious levels of interferon 3 greatly potentiate the TNF1 toxicity. These observations underscore the importance of cell-to-cell communications in regulating pre-OL death pathways. Our preliminary results also suggest that ceramide, a sphingomyelin lipid and a lipid second messenger, acts as a common mediator for pre-OL injury, and that its expression is markedly increased in reactive astrocytes in human PVL. Our overall hypothesis is that activated microglia and astrocytes act corporately in mediating injury to pre-OLs in PVL. We will test the idea that ceramide is a key factor in inflammatory pre-OL death, in part via interactions with TNF1 signaling. Our Specific Aims are to: (1) determine the inhibitory effect of astrocytes on peroxynitrite-induced toxicity to pre-OLs;(2) identify the mechanisms underlying TNF1- mediated pre-OL death;(3) investigate the basis of synergy between the ceramide pathway and TNF1- mediated toxicity;and (4) examine spatial correlations among aberrant upregulation of ceramide, proinflammatory cytokines, and pre-OL cell death in human PVL lesions. We will use various combinations of primary cultures from wildtype and knockout mice to specifically dissect TNF1 signaling in mediating pre-OL death. This project is a significant departure from previous studies in that we focus directly on the molecular and intercellular mechanisms of inflammatory injury to pre-OLs and integrate cell culture studies with human PVL studies. Fundamental insights into such interactions among glial cells could lead to development of novel strategies for the treatment of PVL.PROJECT NARRATIVE White matter injury in preterm infants is a major cause of life-long neurological deficits in survivors of neonatal intensive care;and inflammation appears to play a deleterious role in the injury. Through this proposed study, we hope to reveal how various cells interact and intensify with each other and cause damage to the white matter. Such fundamental insights into the cellular and molecular mechanisms of neonatal white matter injury will provide new avenues for developing novel strategies for the prevention and treatment of this devastating disorder.